Anais da Academia Brasileira de Ciências (2005) 77(3): 549-579 (Annals of the Brazilian Academy of Sciences) ISSN 0001-3765 www.scielo.br/aabc

History on the biological nitrogen fixation research in graminaceous plants: special emphasis on the Brazilian experience

JOSÉ I. BALDANI and VERA L.D. BALDANI

Embrapa Agrobiologia, BR465, Km 07, 23851-970 Seropédica, Rio de Janeiro, Brasil

Manuscript received on December 27, 2004; accepted for publication on April 6, 2005; presented by Eurípedes Malavolta

ABSTRACT This review covers the history on Biological Nitrogen Fixation (BNF) in Graminaceous plants grown in Brazil, and describes research progress made over the last 40 years, most of which was coordinated by Johanna Döbereiner. One notable accomplishment during this period was the discovery of several nitrogen-fixing bac- teria such as the rhizospheric (Beijerinckia fluminensis and Azotobacter paspali), associative (Azospirillum lipoferum, A. brasilense, A. amazonense) and the endophytic (Herbaspirillum seropedicae, H. rubrisub- albicans, Gluconacetobacter diazotrophicus, Burkholderia brasilensis and B. tropica). The role of these diazotrophs in association with grasses, mainly with cereal plants, has been studied and a lot of progress has been achieved in the ecological, physiological, biochemical, and genetic aspects. The mechanisms of colonization and infection of the plant tissues are better understood, and the BNF contribution to the soil/plant system has been determined. Inoculation studies with diazotrophs showed that endophytic bacteria have a much higher BNF contribution potential than associative diazotrophs. In addition, it was found that the plant genotype influences the plant/bacteria association. Recent data suggest that more studies should be conducted on the endophytic association to strengthen the BNF potential. The ongoing genome sequencing programs: RIOGENE (Gluconacetobacter diazotrophicus) and GENOPAR (Herbaspirillum seropedicae) reflect the commitment to the BNF study in Brazil and should allow the country to continue in the forefront of research related to the BNF process in Graminaceous plants. Key words: bacterial endophytes, diazotrophs, semi-solid N-free medium, inoculation, cereals, grass plants.

INTRODUCTION on the occurrence of Azotobacter in acid soils of the “Baixada fluminense” (Döbereiner 1953). These Research on BNF with grasses in Brazil was initiated studies gained visibility with the discovery of two by Johanna Döbereiner when she joined the research new nitrogen-fixing bacteria associated with the rhi- team at the National Center of Education and Agri- zosphere of some gramineous plants: Beijerinckia cultural Research of the Ministry of Agriculture, lo- fluminensis with sugarcane (Döbereiner and Ruschel cated at Km 47 in the fifties. The first studies were 1958) and Azotobacter paspali with Paspalum no-

Dedicated to the memory of Dr. Johanna Döbereiner by two of tatum cv. batatais (Döbereiner 1966). In the sev- her disciples who learned through working with her that research enties, a significant advance in the area of BNF in could be done with simplicity, perseverance, honesty, ethics and grasses was the introduction of the acetylene reduc- sagacity. tion method. Almost at the same time, the semi- Correspondence to: José Ivo Baldani E-mail: [email protected] solid NFb medium that replicated the oxygen level

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found in soil niches was developed to isolate mi- et al. 1997a). New nitrogen-fixing bacteria were croaerophilic nitrogen-fixing bacteria associated identified: Herbaspirillum rubrisubalbicans (Bal- with plant roots. This medium allowed the isolation dani et al. 1996), Herbaspirillum frisingense (Kirch- of two new species of Azospirillum: A. lipoferum hof et al. 2001), Azospirillum doebereinerae (Eck- and A. brasilense. This marked the beginning of ert et al. 2001), Gluconacetobacter johannae and BNF research in grasses in Brazil as well in other Gluconacetobacter azotocaptans (Fuentes-Ramírez countries. The research concentrated on several ar- et al. 2001) and Burkholderia tropica (Reis et al. eas of the interaction between plants and bacteria 2004). including the microorganisms themselves. A new This review aims to rescue the history of BNF species of Azospirillum named A. amazonense studies with grasses in Brazil. These studies were (Magalhães et al. 1983) was isolated and identified mainly coordinated by Johanna Döbereiner and using semi-solid LGI medium, derived from modifi- through her efforts this line of investigation was es- cation of the pH and carbon source of NFb medium tablished in several parts of the World. In addition, (Baldani et al. 1984). it presents recent advances in the area. Several lines of research focusing on agricul- tural applications were developed. Hormonal ef- Free-living and Rhizospheric fects, nitrogen assimilation, biological nitrogen fix- Nitrogen-fixing Bacteria ation and even negative responses were frequently Azotobacter chroococcum observed (Boddey and Döbereiner 1982). During These studies were mostly done between 1950 and this period, other groups working on BNF with non- 1970 when very little was known about the occur- leguminous plants were established in Brazil. Cur- rence of these bacteria in tropical soils. In the first rently, besides the Embrapa Agrobiologia (Km 47) paper on the subject, Döbereiner (1953) observed a team there is groups in the States of Paraná, Rio very frequent occurrence of Azotobacter chroococ- Grande do Sul, Rio de Janeiro, Minas Gerais, Goiás, cum in 22 out of 27 acid soil samples collected in Ceará and Distrito Federal. the “Baixada fluminense”. Research on the colonization of plant tissues by diazotrophic bacteria received a lot of attention from 1985 to 1990 and consequently some aspects of Beijerinckia fluminensis the plant-bacteria interaction began to be elucidated. The occurrence of nitrogen-fixing bacteria of the Two new nitrogen-fixing bacteria able to colonize genus Beijerinckia was mentioned for the first time the interior of plant tissues were found: Herbaspiril- in Brazil and it was demonstrated that the size of lum seropedicae, was isolated from plants of maize, the populations was related to vegetation, physical, sorghum and rice (Baldani et al. 1986a) and Glu- and chemical characteristics of the soil (Döbere- conacetobacter diazotrophicus (synon. Acetobacter iner and Castro 1955). Additional studies on the diazotrophicus) was isolated from sugarcane plants occurrence of this genus in soil of several Brazil- (Cavalcante and Döbereiner 1988). ian States (Rio de Janeiro, São Paulo, Pernambuco This intimate interaction between macro and and Paraná) led to the description of a new species micro symbionts modified the concept known as of Beijerinckia named B. fluminensis (Döbereiner associative and Döbereiner (1992a) introduced the and Ruschel 1958). Analysis of 158 samples col- endophyte concept to the field of BNF. From this lected in different regions of Brazil showed that this point on, a new area within BNF was established species occurred predominantly in soils where sug- which led to great advances in the understanding of arcane was cultivated (Döbereiner 1959a) and a di- physiology, ecology and genetics as well as in the rect influence of the plant on the development of the interaction of the bacteria with the plant (Baldani bacteria was suggested (Döbereiner 1959b). Addi-

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tional studies showed that roots as well as leaves this grass showed that Beijerinckia and Azotobacter and stems had a positive influence on Beijerinckia were the predominant bacteria in the rhizosphere, populations. This was influenced by the exudation even with plants grown in acid soils (Ruschel and of substances into the soil by the roots during rain- Döbereiner 1965). The use of silica gel plates, con- fall (Döbereiner and Alvahydo 1959). Other stud- taining Winogradsky salts and calcium citrate as a ies involving the roots showed that the population carbon source, inoculated with rhizoplane or rhizo- of Beijerinckia was much more pronounced in the sphere soil of Paspalum notatum, led to the isolation rhizoplane region (refers to the soil adherent to the of a new species of Azotobacter, named Azotobac- root surface) than in the rhizosphere. In addition, ter paspali (Döbereiner 1966). This name was later it was shown that removal of the aerial part of the changed to Azorhizophilus paspali (Thompson and plant significantly reduced the population of bacte- Skerman 1981). The high frequency of this bac- ria in both the rhizoplane and rhizosphere regions terium in the rhizoplane of P. notatum (75 out of (Döbereiner 1961). Rice plants grown in green- 76 samples) and in two of three samples of P. plica- house and inoculated with Beijerinckia, showed the tum, as well as its complete absence in 81 rhizoplane establishment of the bacteria as well as an increase samples of other plants including 5 species of Pas- in the yield (Döbereiner and Ruschel 1961). How- palum, led to the suggestion that the observed speci- ever, these types of experiments were not continued. ficity could represent an intermediate stage between Studies on the occurrence of Beijerinckia and Azoto- the legume symbiosis and the non-symbiotic nitro- bacter were extended to other forage grasses and the gen fixation (Döbereiner 1970). Additional stud- results showed that Beijerinckia appears mainly in ies concerning the dependency of the bacteria on rhizoplane soil and Azotobacter in the rhizosphere the plant showed that the bacteria developed much (Ruschel and Döbereiner 1965). better on the rhizoplane (Machado and Döbereiner In the 1970’s, the introduction of acetylene re- 1969). These authors also showed that root exu- duction methodology stimulated further studies in- dates stimulated growth of the bacterial population volving Beijerinckia and sugarcane. The measure- and that active substances present in these exudates ment of the nitrogenase activity in roots of sugarcane tolerated high temperatures. One of the first evalu- showed that it was much higher than that observed in ations on the BNF contribution of A. paspali to the the rhizosphere and in soil between the plant rows. plant was carried out by Donald Kass in 1970, and Beijerinckia indica was the most abundant bacterial he detected a gain of 18 kg N/ha/year (Kass et al. species in both roots and soil samples (Döbereiner 1971). At this time, the acetylene reduction activity et al. 1972a). Quantification of BNF in sugarcane (ARA) technique was being used and one addendum based on the extrapolation of the nitrogenase activ- in the paper mentioned above indicated nitrogenase ity data indicated a contribution of 50kg N/ha/year activities up to 25 nmols C2H4/h/g dry root weight to the soil/plant system (Döbereiner et al. 1973). of batatais grass harvest in the field (Döbereiner and Campêlo 1971). Other studies confirmed the ARA observed for the cultivar batatais and also showed Azotobacter paspali that it was almost zero for the pensacola cultivar A gramineous plant that caught the attention of Jo- (Döbereiner et al. 1972b). The highest activity was hanna Döbereiner in the 1960’s was Paspalum no- observed in the roots and rhizomes, located lower tatum, also known as forquilha or batatais grass. in the soil and was zero in the aerial parts. In addi- Today this grass still covers the campus of Km 47 tion, the authors observed that the bacteria formed and lends the landscape a green color in the absence microcolonies adjacent to roots of cv. batatais but of added nitrogen fertilizer. A preliminary analysis not in the cv. pensacola. Extrapolation of the ARA on the occurrence of nitrogen-fixing bacteria with data to BNF suggested a contribution of up to 50 kg

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N/ha/year (Döbereiner et al. 1973). BNF in cv. Institute, Universidade Federal do Rio de Janeiro. 15 batatais was confirmed by the N2 technique with Nowadays, Dr. Lucy Seldin’s group is continuing the incorporation of 15N into the plant tissues (De- the study of this Gram positive diazotroph. It has Polli et al. 1977). Later, Boddey et al. (1983) used been demonstrated that P. azotofixans is genetically the 15N dilution technique and demonstrated a BNF diverse and that there are a predominance of cer- contribution of 20 kg N/ha/year in plants grown un- tain genotypes of P. azotofixans in the rhizosphere der field conditions. of grasses such as wheat and sugarcane (Rosado et al. 1998). A “rhizosphere effect” promoted by the Derxia spp wheat rhizosphere was also demonstrated (Rosado et al. 1996). A significant difference among the pop- Other nitrogen-fixing bacteria studied by Dr. Jo- ulations of P. azotofixans isolated from rhizoplane, hanna Döbereiner’s group in the 1960’s belonged rhizosphere and soil of maize variety commonly cul- to the genus Derxia, represented by the species D. tivated in Brazil has been observed. In addition, it gummosa and D. indica. A preliminary study on has been shown that the soil type was responsible the occurrence of D. gummosa in soils of Rio de for this diversity (Seldin et al. 1998). These studies Janeiro State, led to the isolation of this bacterium led to the isolation and description of a new species from 20 rhizosphere soil samples collected from dif- of Paenibacillus named P.brasilensis (von der Weid ferent forage grasses (Döbereiner 1968). A more et al. 2002). complete evaluation on the occurrence of Derxia in 100 soil samples from 4 Brazilian States (SP, RJ, PA and PE), cultivated mainly with grasses, showed its ASSOCIATIVE NITROGEN-FIXING BACTERIA occurrence in 36% of root samples collected in RJ Semi-solid Medium and Discovery of Spirillum and PA, but not from other States represented mostly The studies on associative bacteria began with the by samples from very dry regions (Campêlo and use of the acetylene reduction method to measure Döbereiner 1970). A unique experiment on the inoc- the capacity of gramineous plants to fix nitrogen in ulation of Azotobacter vinelandii, Azotobacter pas- association with diazotrophs. The results showed pali, Derxia sp. and Beijerinckia indica in Pennise- high nitrogenase activity rates (ARA), however there tum purpureum plants, grown in greenhouse, was was no direct relation to the nitrogen-fixing bacteria conducted by Souto and Döbereiner (1967). They known at that time. During a talk at the International observed a small but significant increase due to in- Conference on the Global Impact of the Applied oculation of the first three bacteria, however these Microbiology, held in São Paulo, 1973, nitrogen- bacteria did not colonize the rhizosphere. On the free semi-solid medium was mentioned for the first other hand, B. indica significantly increased the dry time. This medium, containing starch or glycerol weight and total N of the plant and also colonized the as a carbon source and calcium carbonate, when rhizosphere. Unfortunately, these grass inoculation inoculated with small pieces of washed roots from studies were not continued. gramineous plants gave rise to the development of an abundant pellicle with high nitrogenase activ- Paenebacillus azotofixans ity. However, it was not possible to isolate and Dr. Johanna Döbereiner also made an initial con- identify the predominant bacteria due to the diffi- tribution to the study of the nitrogen-fixing bac- culties in growing the organisms on plates contain- terium Paenebacillus azotofixans (formerly Bacillus ing nitrogen-free medium, even under low oxygen azotofixans) (Seldin et al. 1984), characterized by levels (Döbereiner 1973). During the first Interna- Dr. Elisa Gastão da Cunha Penido’s group in the tional Congress on BNF, held in Pullman, WA, USA, Microbial Genetic Laboratory of the Microbiology 1974, it was demonstrated that Spirillum lipoferum

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was the main nitrogen-fixing microorganism asso- C2H4/h/g of roots, which varied with the season and ciated with the roots of the forage grass Digitaria stage of plant development (Day et al. 1975). Soil decumbens (Döbereiner and Day 1975). In addi- temperature (Abrantes et al. 1976b) and the level of tion, the authors suggested that this bacterium was ammonia in the soil (Neves et al. 1976) also inter- responsible for the highARA rates detected. Chang- fered with the nitrogenase activity. Due to the po- ing the carbon source of the above-mentioned semi- tential of BNF in grasses, the studies were directed solid medium to sodium malate led to the isolation of towards plants of higher agricultural and economical this microaerophilic bacterium from roots of several importance such as cereals. Values of ARA around gramineous plants. Later studies on the physiology 10,000 nmols C2H4/h/g dry roots were detected in of S. lipoferum showed that growth was pH depen- maize plants grown in pots containing very wet soils dent (best at pH 6.8 to 7.8). The optimum growth and under high light intensity (Dommergues et al. temperature under nitrogen-fixing conditions was 1973). At that time, the authors suggested that anaer- between 32 and 40◦C. Although growth was aer- obic nitrogen-fixing bacteria were the responsible obic, this organism was sensitive to oxygen and the for the activity. In 1975, von Bülow and Döbere- best carbon sources were organic acids (Day and iner published a study on the BNF potential of 276 Döbereiner 1976). Studies on the localization of S. S1 lines of maize. After an ARA pre-screening, 17 lipoferum in roots of D. decumbens, using the tetra- lines were tested under field conditions. The best zolium reduction technique, showed that the bacteria lines continued to show high ARA values (between were mostly found in the inner cortex layer. This led 2,000 and 7,000 nmols C2H4/h/g roots) compared to the suggestion that S. lipoferum was an interme- to 313 nmols C2H4/h/g roots in the original cul- diate between bacteria involved in the rhizospheric tivar. The authors verified the higher ARA activ- association and the legume symbiosis (Döbereiner ity during the flowering stage of maize and were and Day 1975). able to isolate S. lipoferum even after surface ster- ilization of the roots with different agents (alcohol, BNF Grass Potential Measurements by ARA chlorinated water, and hydrogen peroxide). They After the discovery mentioned above, studies were suggested that the association between S. lipoferum carried out to evaluate the BNF potential of several and the roots was located within the root tissues, forage grasses. In addition, it was observed that since the very high ARA could not be explained by there were a few problems in applying the acetylene a simple causal association at the rhizosphere level. reduction technique to samples harvested directly Based on the differences among genotypes, it was from the field. A lag phase was detected during suggested that genetic studies on cereals that are able the measurement of the nitrogenase activity when to associate with diazotrophs should be initiated on extracted roots were used but not when the intact a species other than maize since maize breeding pro- soil/plant system was evaluated. The strategy used grams were mostly directed toward an N fertilizer to solve this problem was to maintain the root sam- response. Therefore, the characteristics that favored ples in flasks containing water and then substitute the association with nitrogen fixing bacteria were it with nitrogen gas in the laboratory. The flasks eliminated in this plant (Döbereiner 1976). were incubated overnight at a low oxygen level be- fore the injection of 10% of acetylene (Abrantes et NFb Medium and Description of al. 1976a). The use of this technique to evaluate the Azospirillum Genus BNF potential of several forage grasses (Panicum At the end of 1975, the semi-solid medium devel- maximum, Pennisetum purpureum, Brachiaria mu- oped for the isolation of Spirillum species was of- tica, Digitaria decumbens, Cynodon dactylon and ficially named NFb (N stands for new and Fb for Melinis minutiflora) gave rates of 239 to 750 nmols Fábio Pedrosa). This medium was used to study the

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occurrence of S. lipoferum in several plants grown in and Döbereiner 1975) and wheat cultivars (Nery et tropical and temperate regions of Brazil and the USA al. 1977). A highly significant negative correlation

(Döbereiner et al. 1976). The results showed that was observed between nitrogenase (N2-ase) in the the S. lipoferum was very common in soil and in the roots and nitrate reductase (NR) in the leaves with roots of plants grown in tropical regions (Brazil and maize lines UR-I selected for high and low BNF.The several African countries). Forage grasses and cere- results suggested that breeding programs focusing als always had a large bacterial population. Despite on BNF should also include studies on NR to select its lower occurrence, S. lipoferum was also isolated genotypes that gain from both N sources (Baldani et from plants grown under non-tropical conditions in al. 1979). the USA and Southern Brazil. A soil pH, between 5.5 and 7.0, favored the occurrence of the bacteria The ARA Method and its Role in the (Döbereiner et al. 1976). Preliminary field inocu- BNF in Grass lation experiments carried out in Madison, Wiscon- Due to its high impact on the study of BNF in sin, USA showed the establishment of the bacteria grasses, the ARA method was the subject of discus- in the roots of the plants. However, at that time the sion by the scientific community. The main topic S. lipoferum inoculation practice in tropical regions referred to the use of isolated roots to measure the did not offer great prospects because of the wide potential of BNF in different cereal genotypes. Mul- distribution of the bacteria in tropical soils. tiplication of bacteria during the incubation of roots Advances in physiology and biochemistry sug- at low oxygen levels was the main criticism. In gested that other aspects should be studied. It was contrast to legumes, nitrogenase activity was de- suggested that inoculation should only be done in the tected only after incubation of the roots for 10 to presence of low doses of nitrogen fertilizers to pre- 16 hours at low oxygen tension. This lag phase vent inhibition/repression of the BNF process and to could, however, be reduced when carbonate was also lessen the environmental risks caused by the ni- added to the roots (Baldani et al. 1978). A posi- trogen fertilizer excess (Döbereiner 1977a). During tive correlation between the nitrogenase activity pro- an International symposium dealing with the appli- duced by pieces of maize and Digitaria roots pressed cation of genetic engineering to the field of nitro- into semi-solid NFb medium and by isolated roots gen fixation, several aspects of the plant/bacteria in- of the same plants was demonstrated by Döbere- teraction were discussed concerning the possibility iner and collaborators (Döbereiner 1978). This sug- of increasing the BNF association in grass plants gested that Spirillum was the main microorganism (Döbereiner 1977b). At the meeting an interesting responsible for the ARA in these plants. Studies comment was made by Dr. Döbereiner. She said that on BNF in 5 tropical forage grasses using isolated the international scientific community did not care- roots showed that the methodology underestimated fully read the papers published on BNF in grasses the ARA values in comparison to those detected us- therefore leading to a race to find S. lipoferum strains ing the intact soil/plant system, although both tech- and maize lines to be used in agriculture. Although niques showed a significant correlation (Souto and the potential of BNF in grasses was already demon- Döbereiner 1984). strated, the studies indicated that the best strategy to maximize BNF was through plant breeding pro- Physiological and Biochemistry Studies grams (Döbereiner 1977c). Several studies carried Several physiological and biochemistry studies were out at that time showed that the plant genotype conducted to gain a better understanding of the role played an important role in the BNF process since played by Spirillum lipoferum in the association with there were highly significant differences in the nitro- grass plants. These studies confirmed observations genase activity observed among maize (von Bülow that the bacteria were sensitive to high levels of oxy-

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gen and that the optimal pH for growth was between Other Azospirillum species – During the last 6.8 and 7.0 and the optimal incubation temperature two decades, other species of Azospirillum have was between 32 and 36◦C. In addition, it was found been described: A. halopraeferens associated with that organic acids were the preferred carbon sources. Kallar grass grown in saline soils of Pakistan Therefore, malic acid was the carbon source incor- (Reinhold et al. 1987), A. irakense associated with porated in the semi-solid NFb medium used to grow rice plants grown in Iraq (Khammas et al. 1989), A. and isolate Spirillum lipoferum (Neyra and Döbere- largimobile (the original name largomobile was or- iner 1977). Another characteristic of the bacteria thographically incorrect) isolated from a water sam- discovered at that time was the ability of some strains ple collected from a lake in Australia (Dekhil et al. to participate in several steps of the nitrogen cycle 1997) and A. doebereinerae associated with Penni- in nature. The most surprising aspect was the abil- setun plants grown in Germany (Eckert et al. 2001). ity to carry out denitrification since nitrogen-fixing Very few studies have been carried out with these bacteria were not known to carry out this process at Azospirillum species, except for A. irakense where that time (Neyra et al. 1977). Based on physiologi- pectinolytic activity was observed (VandeBroek and cal and biochemical characteristics, three groups of Vanderleyden 1995). bacteria were identified: Group I – no biotin require- ment and the ability to use glucose as carbon source Physiological and Biochemical Studies for growth and nitrogen fixation; Group II – a bi- Evaluation of the nitrogen fixation mechanisms in otin requirement and glucose utilization; Group III these Azospirillum species showed that inhibition – similar to Group I, except that it was able to reduce of nitrogenase activity by nitrate was dependent on

NO2- to N2 (Sampaio et al. 1978). the reduction of nitrate (Magalhães et al. 1978). A. lipoferum and A. brasilense species – Later In addition, the authors showed that under aerobic studies, based on DNA:DNA homology, led to the conditions, where nitrogenase is inhibited by oxy- creation of a new genus called Azospirillum with two gen, nitrate could be used as a nitrogen source for species: Azospirillum lipoferum (synon. of Spiril- growth. An elegant schema presented by Döbereiner lum lipoferum) andAzospirillum brasilense (Tarrand (1979) illustrates how oxygen and mineral nitrogen et al. 1978). Serological studies showed that fluores- sources interfere with the BNF process in Azospir- cent antibodies could be used to differentiate the two illum. It was observed that when the oxygen supply species, however 3 subgroups were formed within to the bacterial site exceeds its consumption, NH3,

A. brasilense (De-Polli et al. 1980). NO3 and NO2 are assimilated at maximal rates, but A. amazonense species –A new species ofAzos- there is no BNF. In contrast, when the consump- pirillum, named A. amazonense, was later described tion of oxygen corresponds exactly to the amount (Magalhães et al. 1983). The species was isolated transported to the site, the conditions are optimum from forage grasses grown in the Amazon and in the for nitrogenase synthesis by the bacteria and in the

State of Rio de Janeiro and later from rice, maize case where mineral N sources (NH3,NO3 and NO2) and sorghum plants grown in Seropédica, Rio de are not available, atmospheric N2 is used as nitro- Janeiro (Baldani 1984). Its broad ecological distri- gen source. If oxygen is removed, respiration is bution was later confirmed through the detection of interrupted, and ATP is not generated. This, in turn, large numbers of the bacteria in other grasses such results in the cessation of nitrogen fixation. On the as sugarcane (Baldani et al. 1999). The main char- other hand, nitrate can be substituted by oxygen. acteristics that differentiate it from other species are In this case, the product of respiration (nitrite) is the ability to use sucrose as carbon source, a smaller excluded from the cells. Nitrogen fixation is not in- cell diameter and an inability to tolerate alkaline pHs hibited and the process becomes dependent on (Magalhães et al. 1983). nitrate as demonstrated by Scott et al. (1979). A

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study on the inorganic N transformation processes species known up to 1980, showed that there was a in presence of Azospirillum, showed that the bacte- certain host plant specificity in the colonization of C3 ria participate in all steps except in the nitrification and C4 plants by Azospirillum (Baldani and Döbere- process (Bothe et al. 1981). The authors verified that iner 1980). The authors observed that maize plants nitrogenase activity dependent on nitrate occurs only were preferentially colonized by A. lipoferum while during 3 to 4 hours until the assimilatory enzymes wheat and rice were colonized by A. brasilense. involved in the reduction of nitrate are synthesized. These results were later confirmed for other forage

During this period, nitrite accumulates and the nitro- grasses with C3 and C4 photosynthetic pathways and genase is inhibited when the concentration reaches in addition showed a higher occurrence of denitrify- about 1 mM. Additional studies on the tolerance of ing strains colonizing the interior of roots (Baldani Azospirillum to oxygen carried out in a fermenter, et al. 1981). Another interesting characteristic of showed that the level of tolerance is dependent on the the Azospirillum that colonized the interior of roots age of the culture, optical density and rate of shaking of cereals was the high frequency of isolates with (Volpon et al. 1981). Oxygen tolerance is greater in a tolerance for up to 20 ppm of streptomycin as A. lipoferum and occurred when the concentration compared to strains isolated from the soil or rhi- of lactate and glucose in the medium decreases to zosphere (Döbereiner and Baldani 1979). This dis- less than 0.5% (Stephan et al. 1981, Volpon et al. covery led to a suggestion that the plants developed 1981). More information on the physiology and bio- a new mechanism to select root-colonizing bacteria chemistry of these 3 species of Azospirillum can be (Döbereiner 1979). Further studies showed that lim- found in a book written by Döbereiner and Pedrosa ing stimulated the production of streptomycin in the (1987). rhizoplane of several gramineous plants (Baldani et al. 1982). Ecological and Colonization Studies In the International Workshop on Associative

Several studies were carried out on the coloniza- N2-Fixation, held in 1979, in Piracicaba, SP, Brazil tion process and the establishment of Azospirillum a new term “diazotrophic biocoenosis” was intro- in different grasses. One of the first studies made use duced to describe the association of plants of the of the tetrazolium reduction method (Patriquin and family Gramineae (renamed Poacea) with nitro- Döbereiner 1978). The authors observed bacterial gen-fixing bacteria (De-Polli and Döbereiner 1980). colonization of the cortex tissues and the inner cen- Three more specific terms were also considered; tral region of maize roots and other forage grasses. “rhizocoenosis” (roots), “caulocoenosis” (stems) Further studies, using maize plants grown in the field and “phylocoenosis” (leaves). However, these new confirmed the endorhizospheric nature of the asso- terms were not used by the scientists working in this ciation with bacteria present in the central cylinder area, therefore the term “associative” continued to of the roots as well as in the xylem vessels in colm be used to describe the association of diazotrophic nodal regions (Magalhães et al. 1979). The highest bacteria, mainly Azospirillum, with non-leguminous frequency of colonization occurred during the grain plants. Later 1992a, Döbereiner introduced (as dis- filling stage (105 to 107 cells/g root tissue) when cussed below) the term endophyte, to define bacte- the nitrogenase activity is usually much higher. Al- ria able to colonize internal plant tissues. A group though this methodology and the results have been coordinated by Dr. Fátima Moreira (Universidade subjected to criticism, the intercellular colonization Federal de Lavras, UFLA, Minas Gerais) detected a of maize and wheat plants by Azospirillum has been large population of this species in plants of the Or- confirmed more recently by molecular techniques chidaceae family as well as in species of other plants (Assmus et al. 1995). (Lange and Moreira 2002). Further studies carried A survey on the occurrence of the Azospirillum out by her group on the ecology of Azospirillum in

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heavy metal contaminated area as well as in bauxite mologous and heterologous) in wheat and sorghum mining reclamation area showed that the population plants grown in the field (Baldani et al. 1986b). The of Azospirillum was similar to the one detected in authors observed that homologous strains were pref- non-contaminated agricultural ecosystems, but was erentially located in the interior of roots of wheat drastically reduced in the bauxite mining area. On plants (Sp245 and Sp107) and sorghum (Sp S82) the other hand, the use of several gramineous species while the heterologous strains (Sp 7 and Cd) were in this area promoted a qualitative and quantitative found on the root surface. increase in diazotrophic bacteria populations with Other studies confirmed the inoculation effect values higher than those observed for reference areas of Azospirillum strains on wheat plants grown in (Melloni et al. 2004). The analysis of the nitrogen- the field. It was demonstrated that the observed ef- fixing bacteria present in these areas showed a high fect, mainly with homologous strain Sp 245, was diversity of the diazotrophs including Azospirillum not due to BNF but to an increase in the nitrate re- and Herbaspirillum species as well as other uniden- ductase (NR) activity of the bacteria in the roots tified ones (Nóbrega et al. 2004). (Boddey et al. 1986). The role of bacterial NR in the plant N metabolism was confirmed by inocu- Inoculation Responses lating wheat plants grown under gnotobiotic condi- The advances generated in the 70’s led several re- tions with nitrate reductase negative mutants (Fer- searchers to evaluate the effect of Azospirillum inoc- reira et al. 1987). Besides BNF and its role in the ulation on gramineous plants. The results although plant metabolism, inoculation with Azospirillum can inconsistent, indicated a potential contribution by stimulate plant growth through production of auxins, the BNF process of around 40% of the nitrogen re- gibberellins and citokinins (Hartmann and Zimmer quirement based on observations by groups in Brazil 1994). Some countries, not including Brazil, are and Israel (Boddey and Döbereiner 1982). However, commercially producing inoculants based on Azos- the debate about the principal role played by bacte- pirillum (Baldani et al. 1999). However, practical ria in association with plants still remained. Several application still represents an incognito due to in- authors as summarized by Patriquin et al. (1983) consistent results. demonstrated hormonal effects, biological nitrogen fixation and interference in other processes of the Genetics Studies nitrogen assimilation. Reviews published in the last decade have made a Field wheat inoculation experiments with lote of advances in the physiology, biochemistry Azospirillum strains isolated from sterilized roots and genetics of the Azospirillum species. In ad- of wheat (Sp 245, Sp 107 st), showed a consistent dition, the role of these bacteria in the interaction increase in total plant N however, this was not the with gramineae plants and other non-legume plants case for the heterologous strain Sp 7 (Baldani et al. has been confirmed (Vande Broek and Vanderley- 1983). High correlation (r=0.92) was observed be- den 1995, Bashan and Holguin 1997, Steenhoudt tween N accumulation and the number of Azospir- and Vanderleyden 2000). In Brazil, studies on the illum present in sterilized roots but there was not a organization and regulation of the nif genes in the significant correlation with the number of Azospir- BNF process of the genus Azospirillum have been illum detected in washed roots. A positive inoc- conducted by groups led by Dr. Fábio de Oliveira ulation effect on maize plants was observed when Pedrosa, Universidade Federal do Paraná (UFPR) homologous strains of Azospirillum were compared and Dr. Irene Schrank, Universidade Federal do Rio with strains isolated from other plants (Freitas et al. Grande do Sul (UFRGS). 1982). At this time, studies were initiated to evaluate Dr. Pedrosa’s group played an important the location of different strains of Azospirillum (ho- role in research involving the regulation of BNF in

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Azospirillum brasilense. They isolated the first mu- addition, the group determined the genomic struc- tants with mutations in the regulatory genes nifA ture using pulse-field eletrophoresis of the Azospi- and ntrC that code for the transcription activating rillum genus (Martin-Didonet et al. 2000) and also proteins NifA and NtrC (Pedrosa and Yates 1984). constructed gene fusions using gfp and gusA re- These mutants aided the isolation of the ntrBntrC porter genes to monitor the plant/bacteria interaction operon of A. brasilense by genetic complementation (Ramos et al. 2002). of nifA, nifB genes (Knopik et al. 1991, Machado et The studies coordinated by Dr. Schrank’s al. 1995). This work led to an intensified effort at group, Universidade Federal do Rio Grande do Sul/ the molecular level in order to understand the struc- UFRGS, initially concentrated on the genes that en- tural organization and regulation of the nif genes of code the nitrogenase subunits in the A. brasilense this endophytic diazotrophic bacterium. The work species (Schrank et al. 1987). The nitrogenase on Azospirillum brasilense was initially centered on structural gene operon was completely sequenced the isolation and characterization of regulatory mu- and the gene organization was found to be nifHD- tants able to constitutively fix nitrogen in presence of Korf1Y (Passaglia et al. 1991). Furthermore, the

NH4+ (Machado et al. 1991). These mutants were operons orf2nifusvorf4 (Frazzon and Schrank also able to excrete ammonia, the product of the 1998), nifENXorf3orf5fdxAnifQ (Potrich et al. nitrogen fixation process (Machado et al. 1991, Vi- 2001a) and fixABCX (Irene Shrank, personal com- torino et al. 2001). The group has also dedicated munication) were characterized. The group also considerable effort on studying the regulation of the studied the regulation of the nif operon in nitrogen fixation process in A. brasilense. This reg- A. brasilense. The sequence of the structural gene ulation did not depend on the transcription activat- operon showed the presence of two overlap- ing proteins NtrC, NifA and the Sigma RNA poly- ping UASs, a unique characteristic among all of the merize factor, σ N. The nifA promoter, which does nif operons so far analyzed. In vivo assays showed not show typical structural motifs of the promoters that one UAS (UAS2) has higher activity than UAS1 of promoter dependent proteins, was characterized and that NifA binds to the two muted promoters as a typical σ 70 promoter. Studies on chromoso- (Passaglia et al. 1995). Antibodies specific for A. mal nifA::lacZ fusions and plasmid borne fusions brasilense NifA were also produced (Passaglia et showed that nifA expression of A. brasilense is re- al. 1998). It was demonstrated that the binding pressed by ammonium, the principal effector. Low activity of the purified NifA protein is not altered oxygen tension activates while high oxygen tension even when it is inactivated (presence of oxygen) represses. Other results showed that the repression and it is also able to bind to the nifH gene promoter of nifA expression reaches its maximum level as a when the UASs are mutated (Passaglia et al. 1995). result of a synergistic effect between ammonium and Two mutants with higher nitrogen fixation activity oxygen (Fadel-Picheth et al. 1999). than the wild type were obtained during selection Dr. Fabio’s group recently described NtrX of the nif genes (Araújo et al. 1988). One mutant NtrY as a potential regulator involved in nitrate was characterized and it was shown that the gene metabolism in A. brasilense, however it does not (ORF280) containing the mutation encodes a pre- participate in regulation of nitrogen fixation (Ishida dicted gene product that is homologous to proteins et al. 2002). The role of GlnZ in the reactiva- involved in stress (Revers et al. 2000). More re- tion of dinitrogenase reductase under ammonium- cently Dr. Schrank’s group initiated studies with limiting conditions as well as the requirement of the A. amazonense to determine similarities and differ- PII protein to turn the enzyme off were also im- ence between this species and A. brasilense. Using portant to understanding the control of nitrogenase PCR primers designed for nif or fix gene sequences activity in A. brasilense (Klassen et al. 2001). In of A. brasilense, products from A. amazonense ge-

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nomic DNA were isolated and sequenced from phological and physiological characteristics similar the nifHDK, nifUSV, nifENX and fixABC operons. to the genus Azospirillum, DNA: DNA homology Southern blot hybridization was also carried out us- studies showed that they formed a new genus ing different operon probes against the genome of named Herbaspirillum, thus Azospirillum sero- A. amazonense. These results suggest that the nif/fix pedicae was renamed Herbaspirillum seropedicae operons are present in A. amazonense, however (Baldani et al. 1986a). they exhibited different patterns of restriction en- zyme cleavage. The nitrogenase structural genes, Genetics Studies which are conserved among different bacterial gen- Not much progress was made until the beginning era, are also different in A. brasilense and A. ama- of the 90’s when Dr. Fábio Pedrosa’s group fo- zonense. A conserved region was found in the nifD cused their attention on the organization and regu- gene, but the intergenic region of nifH-D and the lation of the nif genes in this species. They isolated promoter region of nifH were quite different. Also, the nifA, and nifB genes along with the ntrBntrC genes involved in the regulation of nitrogen fixation operon of H. seropedicae by genetic complementa- such as those that encode NifA and PII protein tion of A. brasilense Nif minus mutants (Souza et were isolated and partially sequenced (Potrich et al. 1991a, b, Pedrosa et al. 1997). The nitrogenase al. 2001b). structural genes, other nif genes, and genes involved in the regulation of the nitrogen fixation of H. sero- ENDOPHYTIC BACTERIA pedicae were sequenced. The nif genes were found Endophytic Definition in two regions; region I contains the genes contigu- ous with nifA and nifB, while region II contains The term endophyte was first introduced to the the nifHDKENXorf1orf2orf3 operon (Machado et area of nitrogen fixation research associated with al. 1996, Klassen et al. 1999, Pedrosa et al. 2001). Graminaceous plants by Döbereiner (1992a, b). The The nifQmodABC and fixXC genes are located term was defined by De Bary (1866 cited by Stone 3 kbp upstream (Klassen et al. 1999). The group 1986) and refers to mycotic flora that inhabits studied the motif functions present in the promoter the interior of plant tissues. The term was then ap- region of nifA in detail. Structurally, this region plied to bacteria and was the subject of several con- shows a very high complexity. It has two potential ceptual definitions. In general, the term includes NtrC-binding sites, three NifA sites and one IHF all microorganisms that are able to colonize, dur- (integration host factor) – binding site localized up- ing some portion of their life cycle, the inner tissues stream of an σ N type promoter (–24/–12) (Souza et of plants without causing any apparent damage al. 1991b, Wassem et al. 2000, 2002). In summa- to the host (Petrini 1991). Herbaspirillum, Gluco- rizing, the studies showed that NtrC and RpoN are nacetobacter and Burkholderia are the three essential for expression from the nifA promoter and endophytic nitrogen-fixing bacteria studied by the that IHF positively modulates activation by NtrC Brazilian groups presented below. and acts negatively for activation by NifA (Souza et al. 1991a, b, 1999, 2000, Wassem et al. 2000). In Herbaspirillum seropedicae vivo and in vitro studies of the function and struc- The first nitrogen-fixing bacterium with endophytic ture of the H. seropedicae NifA protein showed that characteristics was isolated in 1984 from the rhizo- this transcription activator responds to external sig- sphere, washed roots and surface sterilized roots of nals such as NH4+ and O2. The N-terminal region maize, sorghum and rice plants and was first named of NifA is involved in auto regulation in re-

Azospirillum seropedicae (Baldani et al. 1984). Al- sponse to the negative regulator NH4+ in concert though this group of bacteria showed several mor- with PII which is essential for activation of NifA in

An Acad Bras Cienc (2005) 77 (3) 560 JOSÉ I. BALDANI and VERA L.D. BALDANI

absence of NH4+ (Monteiro et al. 1999a, b). Dr. Pe- rubrisubalbicans and shows many characteristics drosa’s group also demonstrated that the NifA pro- similar to those of the genus Herbaspirillum tein of H. seropedicae, similar to those from the (Döbereiner et al. 1990, Gillis et al. 1991). It α-Proteobacteria (including A. brasilense), is sen- was shown that it has the ability to fix atmospheric sitive to oxygen. Besides, it requires Fe for in vivo nitrogen and that it could also be reisolated from activity and the interdomain region between the C- sugarcane leaves 60 days after artificial inoculation terminal domain and the central domain is involved of the leaves (Pimentel et al. 1991). Additional in oxygen sensitivity (Monteiro et al. 1999a, Souza physiological studies showed that this bacterium has et al. 1999). In addition, Dr. Pedrosa’s group iso- some characteristics that are different from those of lated and characterized the glnB gene of H. H. seropedicae (Baldani et al. 1992a). Based on seropedicae and determined the three-dimensional this information, a complete study on this group of structure of the purified protein product, a trans- bacteria was carried out and culminated with the ducer protein of the PII signal. This structure is sim- description of a new species named H. rubrisub- ilar to that of the GlnK of enterobacteriaceae and it albicans (Baldani et al. 1996). The genus has re- has a role in the control of NifA activity (Benelli cently been expanded with the description of a new et al. 1997, 2001, 2002). The genes glnAntrBntrC species named H. frisingense, isolated from roots were also sequenced by the group (GenBank acces- and leaves of forage grasses grown in Germany and sion number AF0828730) and constitutes a unique Brazil (Kirchhof et al. 2001). A survey on the oc- operon where transcription is initiated from two currence of this genus showed that the species H. promoter sequences. One promoter is sN dependent seropedicae is found associated with several non- and the other is σ 70 dependent and, both promoters leguminous plants but not in soil in the absence of are located upstream of glnA (Persuhn et al. 2000). plants (Olivares et al. 1996). H. rubrisubalbicans Two genes involved in the SOS repair system, recA has been isolated from many Brazilian sugarcane and recX of H. seropedicae were isolated, se- varieties with no symptoms of the mottled stripe quenced and their functions are being determined disease (Döbereiner 1992b), however its ecological (Steffens et al. 1993). distribution is still unknown. Bacteria belonging to the genus Herbaspirillum were detected in wild and Genomic Studies commercial rice varieties cultivated in Japan, how- ever no relation with known species of Herbaspi- The sequence and annotation of the H. seropedi- rillum could be found (Elbeltagy et al. 2001). cae genome has almost been completed by the One aspect that was a strong consideration for GENOPAR Consortium of Parana State, coordi- inclusion of this genus in the endophytic group is its nated by Dr. Pedrosa. The consortium involves a lower survival in soil (Baldani et al. 1992a). Oli- net of 16 laboratories from Paraná, three from Santa vares et al. (1996) verified the observation that the Catarina, two from Rio de Janeiro and one from Rio recovery of H. seropedicae and H. rubrisubalbicans Grande do Sul. The objective of the project is to gain strains inoculated into soil only occurred in the pres- an understanding of the overall metabolic pathways ence of the host plant. This was probably due to the of H. seropedicae with a view towards increasing release of growth promoter substances in the rhizo- the efficiency of the plant/bacteria association. sphere by the plant. However, Arcanjo et al. (2000) were unable to reisolate strains of Herbaspirillum H. rubrisubalbicans from soil samples collected within sugarcane fields Also in the 90’s, a bacterium responsible for even when sugarcane micropropagated plants were the mottled stripe disease in sugarcane was isolated. used as the trapping host. Similarly, no Herbaspi- This strain belongs to the species Pseudomonas rillum strains could be isolated and analyzed us-

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ing the Immunocapture technique from soil samples tion (Silva et al. 2003). Using cryofracture tech- collected between rows of sugarcane in the field nique it was possible to conclude that the amount (Santos et al., unpublished data). and the polymeric composition of the PHB gran- ules changes depending on the physiological condi- Colonization and Infection Studies tion of the bacteria growing in the medium, based The interaction of H. seropedicae and H. rubrisub- mainly on the status of Nitrogen and Carbon. Un- albicans strains with plants has been extensively der endophytic interaction viewed by TEM after studied through the inoculation of sugarcane and high pressure freezing followed by freezing substitu- rice plants grown under sterile conditions followed tion, Herbaspirillum spp. infected leaves as micro- by microscopic analysis. In rice plants, H. sero- colonies into the lumem of the metaxylem vessel and pedicae has been found to colonize the intercel- as single cell/micro-colony at apoplast. In roots, H. lular spaces near the tip of young roots. Appar- seropedicae colonized cortex apoplast, adhered to ently, the bacteria move intercellularly to the cor- plant cell wall (PCW), such as xylem vessels and tex region, since the cortical cells are where large inside apparently dead vascular parenchyma cells. numbers of bacteria have been detected (Baldani et Plant roots inoculated with the bacteria showed the al. 1992b). More recent results, from studies with bacteria associated with the intercellular root cor- aluminum-tolerant rice cultivars grown under axenic tex. Cryotechniques were able to reveal altered as- and field conditions, confirmed observations that H. pects of all bacteria and PCW at adhesion sites and seropedicae colonizes the interior of roots and the bacterial protrusions could be observed too. With aerial part of the plant (Gyaneshwar et al. 2002). great frequency, the bacteria were observed involved Colm of sorghum plants artificially inoculated with by abundant unknowing electron-dense material and H. rubrisubalbicans showed slight symptoms of the many of vesicular structures coming from plant cell “red stripe” disease in the leaves suggesting an ac- wall were also observed. All substances produced tive translocation of the bacteria (Döbereiner et al. by PCW are clearly stimulated in response to bac- 1994). In contrast, no symptoms were observed with terial presence. These bacterial compounds could the inoculation of H. seropedicae strains. Further stimulate the plant cell not only in relation to N2- studies showed a very high rate of infection of the fixing process but also in process involved other xylem vessels and the formation of a structure orig- plant-growth promotion process (Silva et al. 2004). inating from the plant involving grumes of bacteria (James and Olivares 1998). In the case of sugarcane, Inoculation Response it was observed that the bacteria enter the plant near The potential of cereal plants to respond to inocula- the region of root emergence and invade the vascu- tion with H. seropedicae strains has been evaluated lar tissues by colonizing the parenchymatic cells and by many experiments in the last 15 years. The ini- methaxylem vessels (Olivares et al. 1995). tial results showed no positive response for sorghum Most of the microscopic researches carried out (Pereira et al. 1988) and maize (Pereira and Baldani in Brazil with these endophytic diazotrophic bacte- 1995). In the case of rice, inoculation promoted ria are now coordinated by Dr. Fábio Lopes Olivares a yield increase equivalent to the treatment with (UENF University, Rio de Janeiro State). Besides 40 kg of N/ha (Pereira and Baldani 1995). How- the studies involving endophytic establishment in ever, large advances have been made in the last few sugarcane by Herbaspirillum spp. using conven- years through a highly detailed study based on se- tional microscopy (James et al. 1997, Olivares et lection of Herbaspirillum strains grown in associ- al. 1997), cryotechnique approaches have open- ation with rice plants grown under gnotobiotic ing insights for new ultrastructural aspects of the conditions (Baldani et al. 2000). It was observed bacteria in free state and under endophytic interac- that the response of the rice variety to inoculation

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was dependent on the strain used. In some cases, genus Acetobacter with the creation of the species highly significant increases in phytomass accumu- Acetobacter diazotrophicus, a unique nitrogen- lation were observed with the inoculation of some fixing bacterium of this genus (Gillis et al. 1989). strains, whilst the opposite was observed for others Recently, this species was renamed Gluconaceto- (Baldani et al. 2000). Evaluation of these strains bacter diazotrophicus based on the 16S rDNA se- under greenhouse and field conditions also showed quence and the predominant type of ubiquinone significant increases in yield, however this increase produced (Yamada et al. 1997, 1998). Recently, two depended on the rice cultivar tested (Guimarães et new nitrogen-fixing species of this genus were de- al. 2000). scribed: G. azotocaptans and G. johannae, with the last one in honor of Johanna Döbereiner (Fuentes- Ramírez et al. 2001). Gluconacetobacter diazotrophicus

The other diazotrophic bacterium with endophytic Ecological Studies characteristics and object of intense research is G. diazotrophicus was initially isolated from roots Gluconacetobacter diazotrophicus (synon. Aceto- and colms of sugarcane varieties grown in the North- bacter diazotrophicus). The search for this bac- east (PE and AL) and Southeast (SP, MG) of Brazil terium was initiated with the observation that some (Cavalcante and Döbereiner 1988). It could not be sugarcane varieties could obtain around 60% of detected in soil samples collected between sugar- their nitrogen through biological nitrogen fixation cane rows or in roots of 12 weed plants grown in (Lima et al. 1987). Initially, different semi-solid sugarcane fields nor in grain of saccharine sorghum. media were used. However, the medium contain- However, it could be isolated from washed roots ing ¼ of LGI salt supplemented with 250 mL of and aerial tissues of Pennisetum purpureum cv. sugarcane juice and 25 g of crystal sugar was cho- cameroon plants (Döbereiner et al. 1988). Its occur- sen because G. diazotrophicus formed a very thick rence was confirmed in other Brazilian sugarcane yellow pellicle on the surface of the medium. This areas (Döbereiner et al. 1990) as well as in other medium was suitable for the detection of bacteria countries that produce sugarcane such as Australia in root and colm samples up to 107 dilution (Caval- and USA (Baldani et al. 2002a). cante and Döbereiner 1988). Through knowledge Until quite recently, it was thought that G. acquired on the physiology of this organism, the au- diazotrophicus could only occur in plants that prop- thors modified the semi-solid medium, now named agate vegetatively such as sugarcane, sweet potato LGIP, and established as a rule the use of 10% crys- (Paula et al. 1990, Döbereiner et al. 1993) and tal sugar with the pH adjusted to 5.5 using acetic pineapple (Tapia-Hernandez et al. 2000). However, acid. The medium was again modified by Reis et al. it was detected in plants propagated by seeds such (1994) with the addition of sugarcane juice in the as the grass Eleusine coracana (Loganathan et al. concentration of 0.5%. 1999) and in coffee (Fuentes-Ramirez et al. 2001), Based on morphological, physiological and although these observations refer to the new species biochemical characteristics of this group of bacte- mentioned above, G. johannae and G. azotocaptans. ria, a new genus named Saccharobacter was created G. diazotrophicus has already been detected in with a new species called S. nitrocaptans (Caval- mealybugs that inhabit sugarcane fields (Ashbolt cante and Döbereiner 1988). However, DNA: DNA and Inkerman 1990) and in mycorriza spore fungi and rRNA hybridization analysis showed that this collected from sweet potato roots (Paula et al. 1991). group of bacteria belonged to an rRNA branch of Genetic diversity studies conducted on G. diazotro- Acetobacter with many characteristics similar to A. phicus strains isolated from sugarcane plants culti- liquefaciens. Therefore, it was included in the vated in Brazil and Mexico showed that diversity

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is very limited (Caballero-Mellado and Martinez- The optimum pH for its growth is around 5.5 and Romero 1994). Studies from our laboratory with one interesting characteristic is the absence of the 45 strains isolated from sugarcane varieties, main- nitrate reductase enzyme (Cavalcante and Döbere- tained for 30 years in a germoplasma bank and an- iner 1988). Nitrogenase activity is not inhibited or alyzed by the ITS/RFLP technique, also confirmed suppressed by high concentrations (25 mM) of ni- this low genetic diversity (Santos et al. 1999). trate (Stephan et al. 1991), but is partially inhibited

Other studies using ELISA, Immunocapture by NH4+ (Teixeira et al. 1987). Reis and Döbereiner and PCR techniques have been conducted in an ef- (1998) demonstrated that the nitrogenase activity is fort to detect G. diazotrophicus in soil samples and less inhibited by NH4Cl (5 mM) when the bacte- plant tissues. Using the ELISA technique, large ria are grown in media containing 10% sucrose as populations of G. diazotrophicus were detected in compared to 1%. Studies involving a mixed culture different plant tissues of sugarcane varieties culti- of G. diazotrophicus and amylolytic yeast showed vated in Brazil and Australia (Boddey et al. 2000). that G. diazotrophicus is able to support growth of In the case of the Immunocapture technique it was the yeast with about 40% of the fixed nitrogen being possible to detect G. diazotrophicus in plant tissues used by this organism (Cojho et al. 1993). The form but not in soil collected between rows of sugarcane of nitrogen excreted by G. diazotrophicus during plants grown in the field (Santos et al., unpublished the biological nitrogen fixation process is currently data). With the PCR technique, fragments of the unknown (Baldani et al. 1997a). same size as those from G. diazotrophicus genomic DNA were detected in soil samples collected in a Plant Dependence sugarcane field. However, G. diazotrophicus could The dependence of the G. diazotrophicus on not be reisolated from micropropagated sugarcane plants, the majority of which propagate veg- plants used as a trapping host (Arcanjo et al. 2000). etatively (setts-sugarcane and Pennisetum, stem These results suggest that the bacteria could be in a cutting-sweet potato), emphasizes its endophytic viable but non cultivable stage or that the number of life style and suggests that this is the major way of the bacteria in the soil was too low to colonize the dissemination of this organism. Paula et al. (1991) roots of the plants. demonstrated that micropropagated sugarcane and sweet potato showed high colonization of the aerial Physiological and Biochemical Studies part by G. diazotrophicus when co-inoculated with The physiological and biochemical studies carried mycorrizal fungi. Thermal treatment of sugarcane out during the last 15 years on G. diazotrophicus setts (30 minutes at 52◦C), applied to eliminate phy- showed that this bacterium is able to grow diazo- topathogenic bacteria responsible for the ratoon trophically using different carbon sources in a cul- stanting disease, did not eliminate the endophyte ture medium with a pH much lower than that used (Reis et al. 1994), confirming that the setts are one for most of the diazotrophic bacteria (Cavalcante way of dissemination of the bacteria. In addition, it and Döbereiner 1988). G. diazotrophicus is able to has been observed that the bacteria have a very low grow at pH 3.0 and fixes nitrogen in culture medium rate of survival in soil (Baldani et al. 1997a), al- at pH 2.5 (Stephan et al. 1991). This bacterium though it has been demonstrated that the rate of cell does not use sucrose directly – it has an extracel- death depends on the humidity at the time of inocu- lular enzyme (invertase) with saccharolytic activity. lation into the soil (Oliveira et al. 2004). It is already This carbon source is a key compound during the known that the bacterial population is also influ- isolation of the bacteria since the LGIP semi-solid enced by the nutritional stage of the sugarcane plants medium contains 10% sucrose (crystal sugar) which (Reis-Jr et al. 2000) and that nitrogen fertilization is known to inhibit the growth of other diazotrophs. also decreases the population of G. diazotrophicus

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associated with the sugarcane (Fuentes-Ramírez setts. Nevertheless, inoculation of micropropagated et al. 1999). sugarcane plants with the G. diazotrophicus strain PAL5 increased the aerial fresh weight of the plant Genetics Studies by 28% (Baldani et al. 1999). Similar results were In the 90’s, many genes involved in nitrogen fixation obtained when the inoculation of the strain PAL5 by G. diazotrophicus were cloned and sequenced was done in the presence of low amounts of nitrogen (Sevilla et al. 1997, Teixeira et al. 1999). The or- fertilizer (Moraes and Tauk-Tornisielo 1997). Proof ganization of the nif, fix and mop genes is known, that G. diazotrophicus fixes nitrogen was demon- however the mechanisms involved in their regulation strated by inoculation of micropropagated sugar- still needs further study (Lee et al. 2000). Because cane plants with wild-type strain PAL5 and its nif 15 of its endophytic life style, this bacterium has been minus mutant followed by measurement of N2 used as a vector to express heterologous genes of in- gas incorporated into the plant tissues (Sevilla et al. terest. One example is the expression of cry3A and 2001). Recent results have shown that sugarcane cry1Ab genes isolated from Bacillus thuringiensis derives benefit from inoculation with a mixture of that are responsible for the entomopathogenic ac- diazotrophic bacteria (Oliveira et al. 2002). This re- tivity against coleopteran and lepdopeteran insects sult was also obtained when the inoculation process that cause damage to sugarcane plants (Salles et al. is associated with mycorrizae (Muthukumarasamy 2000, Baldani et al. 2002b). et al. 1999). Other effects of G. diazotrophicus inoculation are related to phyto-hormone produc- Inoculation Response tion (indol-acetic acid) which acts on initial root de- One way to introduce G. diazotrophicus into sugar- velopment which in turn benefits the whole plant cane plants is during the micropropagation process as demonstrated by Fuentes-Ramírez et al. (1993). since the setts are naturally colonized by this bac- Other studies of co-inoculation involving G. dia- terium. The micropropagation process eliminates zotrophicus and mycorrizal fungi showed an incre- both the phytopatogenic and the nitrogen-fixing ment in the root system of sweet potato plants as bacteria. Therefore, a method to introduce selected well as the uptake of some nutrients (Paula et al. endophytic diazotrophic strains during the acclima- 1992). The BNF contribution to sugarcane plants tization process was developed (Reis et al. 1999). grown in the field for 18 months after inoculation The bacteria are able to infect and colonize the root at the micropropagated stage with a mixture of en- tissues and aerial parts by penetrating root tip and lat- dophytic diazotrophic bacteria, was in the range of eral roots formed during the rooting process (James 20 to 30% of the total N accumulated in the plant et al. 1994). At the colm base, where the tissues are tissues (Oliveira et al. 2003). Other measurement heavily colonized, the bacteria move to the aerial studies using the delta 15 technique showed that the part using the xylematic vessels (James et al. 1994, average BNF contribution to the sugarcane varieties 2001). Relatively large populations of the bacteria harvested in sugarcane producing areas in Brazil is are found in these tissues, suggesting that this is one around 30% (Boddey et al. 2001). However, these of the main sites for nitrogen fixation due to the low values varied from 0 to 70% depending on the level oxygen level and the energy available in form of of the molybdenum available in the soil (Polidoro sucrose for nitrogen fixation (James et al. 1994). 2001). Few inoculation studies have been done to evaluate the potential of G. diazotrophicus strains to Anatomical and Physiological Root fix nitrogen in association with sugarcane with con- Response to Inoculation tributions to the plant’s nitrogen metabolism, be- The group coordinated by Dr. Olivares has carried cause sugarcane plants are mostly propagated by out studies on the inoculation effect of this

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bacterium on physiological changes of the plant first time that sugarcane might be actively involved metabolism. The authors have demonstrated that in the association, because several genes involved in the inoculation of different micropropagated sugar- different plant physiological processes were identi- cane genotypes (mainly RB cultivars) with selected fied as candidates to be differentially expressed dur- strains of G. diazotrophicus render a sort of anatom- ing the association (Nogueira et al. 2001). The group ical and physiological changes over the plant host. focus now the studies on the characterization of These effects included an increase of lateral root signaling pathways by which sugarcane plants can mitotic sites as well as emerged lateral roots with decipher bacterial signals and respond properly for a changes at the root geometry by increasing the fine successful association (Vargas et al. 2003); and the root portion and the overall root system (Olivares et molecular mechanisms that promote plant growth al. 2002). Futhermore, these anatomical changes by association with the endophytes. By using func- have been accomplished by an increase on the H+- tional genomic tools, a group of genes related to ATPase activity of the root cell microsomal fraction nitrogen metabolism and plant development are be- and protein contents. Besides that, studies were car- ing characterized, in order to understand how plants ried out to link the endophytic inoculation and pho- benefit by this association. In addition, receptors tosyntetic processes. The results showed that the involved in signaling plant/bacteria interactions are net phosynthesis, stomacal conductance, transpira- also being studied. The data observed for most of tion rates as well as the relative quantum dependence the studied genes indicate that the modulated gene of photosystem II are not affected by the endophytic expression during association is not a general stress establishment of Herbaspirillum/G. diazotrophicus, response against microorganisms, but seems to be indicating no photoinhibitor effect and no altered specific for benefic associations. Interestingly, the leaf gas exchange. The physiological changes that data showed that expression of several genes is not takes place in sugarcane during the endophytic inter- altered in sugarcane genotypes with low contribu- action could be, in part, related to the plant growth tions of BNF, indicating that the plant genotype promoting effects such as it has been observed in has an important role on the efficiency of the asso- many experiments that includes increase of nutrient ciation. accumulation and biomass. Genomic and Proteomic Studies Molecular Mechanism of the Plant-bacteria The economical potential for the use of G. diazotro- Interaction phicus for the inoculation of sugarcane cultivated An important feature of the plant interaction with in the Rio de Janeiro State and Brazil stimulated these endophytes is that bacteria colonize most the creation of a network to sequence the genome plant organs, promoting plant growth without caus- of this bacterium. The collaborative network called ing any disease symptoms. It raises the question if RIOGENE is supported by FAPERJ and CNPq and there is an active role of the plant in the process, or has the participation of Embrapa Agrobiologia, four if it is just a niche for bacterial growth. Since 1994, universities (UFRJ, UFRRJ, UENF and UERJ) and Dr. Hemerly’s group from the UFRJ is address- Laboratório Nacional de Computação Científica ing this question by investigating sugarcane gene (LNCC). The aim of the project is to obtain the expression during the association with Gluconace- genome sequence by the end of this year with a tobacter diazotrophicus using different approaches: goal to understand gene functions and consequently (i) cDNA–AFLP fingerprinting, (ii) transcriptional their manipulation to increase the efficiency of the profiles generated from the SUCEST (Sugarcane plant/bacteria interaction and nitrogen fixation. A EST Sequencing Project) database and (iii) microar- proteomic network was created to support the se- ray. The novelty of this studies was to show for the quencing program of G. diazotrophicus.

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Burkholderia spp al. 1997, Reis et al. 2000). Recent studies showed that the B. brasilensis strain M130 and the B. ku- The last group of diazotrophic bacteria showing ruriensis strain KP23 showed the same ARDRA endophytic characteristics, but not as well studied pattern (Santos et al. 2001) with a similarity of as those described above, was renamed in the last 99.9% between the 16S rDNA subunits, suggesting decade (Yabuuchi et al. 1992). This new genus that these two strains belong to the same species. called Burkholderia consists of 47 species, how- Partial sequencing of the nifH and glnB genes also ever only three are known to fix nitrogen (Gillis showed that these strains (M130 and KP23) were et al. 1995, Zhang et al. 2000, Reis et al. 2004). similar but distinct from the reference PPe8 strain The first, named B. vietnamiensis, was isolated belonging to the B. tropicalis species (Marin et al. from the rhizosphere of rice roots cultivated in the 2003). DNA: DNA experiments are been carried Vietnam (Gillis et al. 1995). This nitrogen-fixing out to define either the B. brasilensis isolates consti- species also included two strains isolated from hu- tute a new species or belongs to the B. kururiensis man materials belonging to the older Pseudomonas species. On the other hand, the proposed name of B. cepacea species, demonstrating that the new genus tropicalis was offically accepted as B. tropica (Reis is not restricted to plant materials. The other species et al. 2004). called B. kururiensis was isolated in Japan from an aquifer area contaminated with trichloroethylene Morphological and Physiological Studies (TCE) (Zhang et al. 2000). The ability of this Among the morphological and physiological char- species, represented only by one isolate, to fix ni- acteristics that distinguish the two species are: pH trogen was demonstrated by Santos et al. (2001) in tolerance, use of carbon sources, colony type grown a study that compared different Burkholderia in JMV medium containing nalidixic acid, and os- strains isolated from plants grown in Mexico and motic tolerance. The “B. brasilensis” species grows from other countries including Brazil. and fixes nitrogen in semi-solid JMV medium with In the mid 90’s, a large number of nitrogen- a pH range of 4.0 to 6.0 with mannitol or +D,L- fixing bacteria, with characteristics similar to those carnitine as carbon sources. This strain, however, of the Burkholderia genus, were isolated from rice, does not grow in semi-solid LGIP medium with sugarcane and sweet potato plants using semi-solid 10% sucrose. The colonies in semi-solid JMV JMV medium containing mannitol as a carbon medium are brownish and become irregular, smooth source with the pH adjusted to near 4.5 (Baldani and transparent in the presence of nalidixic acid. 1996). The partial sequence of the 23S and 16S “B. brasilensis” strains are able to oxidize the rDNA region of two representative strains (M130 following carbon sources (Biolog test): maltose and and Ppe8) indicated that these isolates belong to xylitol but not D-lactose and L-raffinose. On the the genus Burkholderia, but they were not B. viet- other hand, the B. tropica species grows and namiensis species. Later, studies using probes de- fixes nitrogen in semi-solid JMV medium with a pH rived from the sequenced regions from M130 and higher than 5.0 with one of the following carbon Ppe8 showed the presence of two distinct groups, sources: mannitol, +arginine, +adipate or +ribose. one formed by the rice, manhiot and sweet potato It is able to fix nitrogen in semi-solid JMV medium isolates and the other by the isolates from sugarcane with 10% of sucrose. Colonies grown on Potato (Hartmann et al. 1995). medium with 10% crystal sugar are light brown and Burkholderia tropica – Based on the morpho- when grown in LGIP medium the colonies are or- logical, physiological and genetic characteristics, ange with brownish borders. Colonies in JMV two new species were proposed: B. brasilensis medium (pH 5.5) containing 2.5 μg/L of nalidixic (Baldani et al. 1997b) and B. tropicalis (Kirchhof et acid are rounded, and orange with yellow borders.

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B. tropica strains are able to oxidize the follow- Inoculation Responses ing carbon sources (Biolog test): D-lactose and L- Several inoculation experiments have been carried raffinose, but not maltose and xylitol. Strains from out to determine the BNF contribution to gramina- both species can be identified through the ceous plants by these Burkholderia species. Rice hybridization with probes based on the sequences varieties grown in low fertility acid soil in Viet- of the 16S ribosomal subunits. The “B. brasilen- nam inoculated with the Burkholderia vietnamien- sis” species can be identified using the Bbra62 and sis TVV75 strain gave yield increases of 13 to 22% Bbra636 probes while the B. tropica species can be (Tran Van et al. 2000). In Brazil, a detailed identified using the Btrop636 and Btrop463 probes study has been conducted involving strains of (Boddey 2003). “Burhkolderia brasilensis” and different rice vari- eties with the goal to select those strains that are more efficient in the association leading to a greater Ecological Studies contribution to the development of the plant (Bal- dani 1996). The results show that an interaction Studies on the ecological distribution of these exists between the strain and rice cultivar (Baldani species demonstrated their high frequency of oc- et al. 2000). A BNF contribution on the order of 20 currence (especially “B. brasilensis”) in sugarcane and 30%, as determined by the 15N isotopic dilution plants grown in different regions of Brazil and in technique was observed in rice plants grown under Australia (Boddey 2003). “B. brasilensis” has also gnotobiotic and greenhouse conditions (Baldani et been isolated from different rice varieties grown in al. 2000). The yield response of the same rice cul- soil from Rio de Janeiro State and the Cerrado soil tivars grown in the field and inoculated with these from Goiás (Rodrigues et al. 2001). Strains from strains was very variable (Guimarães et al. 2000). these two species were also isolated from banana and A yield increase of 54% was observed for the pineapple (Weber et al. 1999) and their identity was IAC4440 rice variety inoculated with “Burkholde- confirmed by the partial sequencing of the16S rDNA ria brasilensis” strain M209, however the increase subunit (Cruz et al. 2001). The infection and colo- was very low for the IR42 variety inoculated with nization process of rice by “Burkholderia brasilen- this strain (Guimarães et al. 2002). The results sug- sis” strain M209 showed that the bacteria first col- gest that BNF research on rice should be increased onize the root surface and than penetrate the cells considering the progress that has been made. via the intercellular spaces of the damaged mem- One aspect that should be exploited should be the brane (Baldani et al. 1997a). The bacteria can also plant/bacteria interaction as well as interactions be- penetrate through wounds in the epidermal cell re- tween bacteria as has been observed for sugarcane gion and points of emergence in the secondary roots inoculated with a mixture of bacteria (Oliveira (Baldani et al. 1995). Additional studies showed et al. 2002). that these species also colonize the stomata of rice seedlings (Silva et al. 2000). The infection and PERSPECTIVES colonization process of micropropageted sugarcane roots inoculated with the strain Ppe8 of Burkholde- A historical analysis of studies on BNF in Grami- ria tropica was very similar to that observed naceous plants demonstrates significant advances in for other endophytic bacteria (Boddey et al. 1999). several aspects of plant/bacteria interactions. How- Large populations of strain Ppe8 are mainly found ever, the expectation that the nitrogen fixation effi- on the surface of micropropagated sugarcane roots, ciency might be equivalent to the rhizobia /legume when this strain is inoculated together other endo- symbiosis did not turn out to be true, although the phytic diazotrophic bacteria (Oliveira et al. 2002). endophytic diazotrophic bacteria/plant association

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shows some characteristics that are similar to the (Herbaspirillum seropedicae, H. rubrisubalbicans, Glu- legume symbiosis. A biotechnological program de- conacetobacter diazotrophicus, Burkholderia brasilensis voted to defining the functionality of genes present e B. tropica). O papel destas bactérias diazotróficas em in most of the nitrogen-fixing bacteria as well as associação com as gramíneas, especialmente os cereais, knowledge generated by genome sequences of sev- tem sido estudado e muito se avançou sobre os aspec- tos ecológicos, fisiológicos, bioquímicos e genéticos. Os eral plants of agronomic interest, should contribute mecanismos de colonização e infecção dos tecidos das to a better understanding of these associations, par- plantas foram melhor entendidos e a contribuição da FBN ticularly the endophytic ones. Consequently, it may para o sistema solo-planta foi determinado. Estudos de be feasible to convert the potential of this associa- inoculação de cereais com bactérias diazotróficas, têm tion into a standard inoculation practice in agricul- mostrado que as endofíticas têm um maior potencial de ture. However, responses similar to those observed contribuição da FBN e que o genótipo da planta influencia for the Brazilian soybean should not be expected for na associação da planta/bactéria. Os avanços alcançados cereals and other grasses inoculated with endophytic apontam para uma maior exploração e entendimento desta diazotrophic bacteria. As has been emphasized in associação endofítica. Os programas de sequenciamento most of Dr Johanna Döbereiner’s papers, breeding do genoma: RIOGENE (Gluconacetobacter diazotrophi- programs with Graminaceous plants should always cus) e GENOPAR (Herbaspirillum seropedicae) mostram a importância da FBN no Brasil e devem permitir que o take the interaction of endophytic diazotrophic bac- país continue na fronteira do conhecimento em relação ao teria and plant genotype into account so that the bi- processo de FBN em gramíneas. ological nitrogen fixation process can be optimized. Palavras-chave: bactéria endofítica, diazotrófica, meio semi-sólido, inoculação, cereais, planta forrageira. ACKNOWLEDGMENTS The authors thank Dr. Fábio Pedrosa, Dr. Irene REFERENCES Schrank, Dr. Lucy Seldin, Dr. Fátima Moreira, Dr. Adriana Hemerly and Dr. Fábio Lopes Olivares Abrantes GTV, Day JM, Cruz VF and Döbereiner J. for providing information that helped to enrich this 1976a. Métodos para o estudo da atividade da nitro- review dedicated to the memory of Jöhanna Döbe- genase em raízes de gramíneas colhidas no campo. In: Congresso Brasileiro de Ciência do Solo, reiner. Thanks also go to Dr. Paul Bishop for review- 15, Sociedade Brasileira de Ciência do Solo, Campi- ing the manuscript and to the Conselho Nacional nas, SP, Brasil, p. 137–142. de Desenvolvimento Científico e Tecnológico/Pro- Abrantes GTV, Day JM, Cruz VF and Döbereiner grama de Apoio a Núcleos de Excelência (CNPq/ J. 1976b. Fatores limitantes da fixação de nitrogênio Pronex II) for financial support of the research car- em campo de Digitaria decumbens v. transvala. In: ried out by the group of Excellence on Biological Congresso Brasileiro de Ciência do Solo, 15, Nitrogen Fixation in Non-leguminous Plants. Sociedade Brasileira de Ciência do Solo, Campinas, SP, Brasil, p. 171–176. RESUMO Araújo EF, Zaha A, Schrank IS and Santos DS. A presente revisão aborda a história da Fixação Bioló- 1988. Characterization of DNA segments adjacents gica de Nitrogênio (FBN) em Gramíneas no Brasil, procu- to the nifHDK genes of Azospirillum brasilense Sp7 rando mostrar a evolução da pesquisa na área iniciada a by Tn5 site-directed mutagenesis. In: Klingmüller mais de 40 anos sob a liderança da pesquisadora Johanna W (Ed), Azospirillum IV: Genetics, Physiology and Döbereiner. Um aspecto marcante deste período foi a Ecology. Berlin: Springer Verlag, p. 16–25. descoberta de diversas bactérias fixadoras de nitrogênio Arcanjo SS, Santos ST, Teixeira KRS and Baldani atmosférico tais com as rizosféricas (Beijerinckia flumi- JI. 2000. Occurrence and dissemination of endo- nensis e Azotobacter paspali), associativas (Azospirillum phytic diazotrophic bacteria in sugarcane fields. In: lipoferum, A. brasilense, A. amazonense) e as endofíticas Pedrosa FO, Hungria M, Yates G and Newton

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